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OPEN A revisited history of cacao

domestication in pre‑Columbian
times revealed by archaeogenomic
approaches
Claire Lanaud 1,2,17*, Hélène Vignes 1,2,17, José Utge 3, Gilles Valette 4, Bénédicte Rhoné 1,2,
Mariella Garcia Caputi 5, Natalia Sofía Angarita Nieto 6, Olivier Fouet 1,2,
Nilesh Gaikwad 7, Sonia Zarrillo 8, Terry G. Powis 9, Ann Cyphers 10, Francisco Valdez 11,
S. Quirino Olivera Nunez 12, Camilla Speller 8, Michael Blake 8, Fred Jr. Valdez 13,
Scott Raymond 14, Sarah M. Rowe 15, Guy S. Duke 15, Francisco Ernesto Romano 6,
Rey Gaston Loor Solórzano 16 & Xavier Argout 1,2

Humans have a long history of transporting and trading plants, contributing to the evolution of
domesticated plants. Theobroma cacao originated in the Neotropics from South America. However,
little is known about its domestication and use in these regions. In this study, ceramic residues from
a large sample of pre-Columbian cultures from South and Central America were analyzed using
archaeogenomic and biochemical approaches. Here we show, for the first time, the widespread use
of cacao in South America out of its native Amazonian area of origin, extending back 5000 years,
likely supported by cultural interactions between the Amazon and the Pacific coast. We observed
that strong genetic mixing between geographically distant cacao populations occurred as early as
the middle Holocene, in South America, driven by humans, favoring the adaptation of T. cacao to
new environments. This complex history of cacao domestication is the basis of today’s cacao tree
populations and its knowledge can help us better manage their genetic resources.

The current diversity of a crop species reflects its past history where the joint impacts of environmental changes
and human-plant interactions have shaped it over centuries and ­millennia1. Through human migrations and
trading routes, a new diversity may emerge in a plant species resulting from exchanges, selection and genetic
mixing between distant and differentiated populations. Humans have a long history of transporting and trading
plants, thereby moving them out of their natural ranges and selecting varieties with traits that are of greatest
interest. Theobroma cacao L., a greek term meaning: “the food of the gods," is one such plant that was of great
economic and symbolic interest to the ancient farmers in the New World. It originated from tropical and humid
regions of South America with hotspots of diversity observed in the upper Amazon basin close to the frontiers
between Colombia and ­Ecuador1–4. This diversity has been classified in ten genetic groups, (Amelonado,
Contamana, Criollo, Curaray, Guiana, Iquitos, Marañon, Nacional, Nanay, and Purús)2, and more recently
classified in eleven genetics groups, with the addition of a supplementary group located in Colombia and named

1
CIRAD, AGAP Institut, Avenue Agropolis, F‑34398 Montpellier, France. 2AGAP Institut, Université de Montpellier,
CIRAD, INRAE, Institut Agro, Montpellier, France. 3UMR 7206 Eco-anthropologie, Département Homme et
Environnement, MNHN-CNRS-Université Paris Cité, Paris, France. 4Institut des Biomolécules Max Mousseron
– (UMR IBMM), Université de Montpellier, Montpellier, France. 5Museo Antropologico y de Arte Contemporaneo
(MAAC), Guayaquil, Ecuador. 6Museo Nacional de Colombia (MNC), Bogotá, Colombia. 7Gaikwad Steroidomics Lab
LLC, Davis, USA. 8Department of Anthropology, University of British Columbia, Vancouver, Canada. 9Department
of Geography and Anthropology, Kennesaw State University, Kennesaw, USA. 10Universidad Nacional Autónoma
de México (UNAM), México, México. 11Institut de Recherche pour le Développement (IRD), UMR 208 PALOC,
MNHN-IRD, Paris, France. 12Asociación para la Investigación Científica de la Amazonía de Perú (ASICAMPE), Lima,
Perú. 13The University of Texas at Austin, Austin, USA. 14Department of Anthropology and Archaeology, University
of Calgary, Calgary, Canada. 15The University of Texas Rio Grande Valley, Edinburg, TX, USA. 16Instituto Nacional de
Investigaciones Agropecuarias, (INIAP), EET Pichilingue, Quevedo, Ecuador. 17These authors contributed equally:
Claire Lanaud and Hélène Vignes. *email: lanaud@cirad.fr; claire.lanaud@gmail.com

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“Caqueta”4. However, it is in Mesoamerica and Central America that the ancient domestication of T. cacao has
been particularly well documented, in part owing to the tree’s cultural significance to the ancient and current
cultures of these regions. Archaeological evidence has shown its economic, social, and cultural importance in
Mokaya, Olmec, and Maya p ­ opulations5–12, and a fine flavor aromatic variety, the Criollo variety, was considered
as the unique variety cultivated in Mesoamerica and Central America. Indeed, in popular culture, as for early
crop ­scientists13, Mesoamerica and Central America are considered the homeland of cacao, even though it was
introduced to the region through past human-mediated d ­ ispersal7,14–17.
Traces of the use and domestication of cacao in South America, dating back to 5300 years BP, have been
documented in the Southern Ecuadorian ­Amazon18 where one of the three ancestors of another fine flavor
aromatic variety, presently cultivated along the Ecuadorian Pacific coast, the Nacional variety, o ­ riginated19,20.
The modern Nacional variety is a hybrid population also involving Criollo and Amelonado ancestors, the latter
of which is an old variety that was cultivated widely in Brazil during the last centuries and where it is thought
to have been domesticated during the eighteenth c­ entury21. The presumed origin of modern Nacional variety
would have resulted from a first introduction of Nacional genotypes, introduced from southeastern Ecuador to
the Pacific coast, followed by the introduction, only a century ago, of Trinitario types (hybrids between Criollo
and Amelonado) from ­Venezuela22.
However, many questions and uncertainties remain about the timing, way of migration and population
diversity of Criollo and Nacional during the domestication steps. Moreover, no new information regarding
cacao’s use during pre-Columbian times has come to light so far, in South America since our previous w ­ ork18.
Our goals are now to trace the migration and use of T. cacao in South America from the Amazonia, its region
of origin, to the Pacific coast where it was introduced and observed by the Spanish upon their arrival on the
Ecuadorian coast. To this end, the presence and ancestry of T. cacao in ceramics from a wide range of human
cultures, present in South America and spanning several millennia, from the earliest ceramic-making peoples
who inhabited the Pacific coast of South America, have been studied. The analysis of ancient DNA (aDNA)
from old remains of plants can be used to study and directly observe past genetic diversity of plant species of
interest, thereby helping us to unravel the history of their d ­ omestication23,24. In this work, no archaeobotanical
cacao remains could be found, but we analyzed the aDNA collected in the ceramic residues of 352 archaeological
items from several pre-Columbian cultures located along the Pacific coasts of Ecuador and Colombia, as well
as in Central America, and dating within the past five millennia. These residues corresponded to food residues
adhered or adsorbed by the ceramic walls. The recent genetic re-sequencing of a collection of modern cacao trees
representing the global diversity of the s­ pecies25 makes it possible for us to analyze, with SNP (Single Nucleotide
Polymorphism) markers, the genetic structure and relatedness between the varieties formerly consumed and
the modern native populations of cacao that originated from various geographical regions. Thus, these analyses
provide clues to the origin of the old varieties and the pathways of their ancient domestication. Our analysis
of aDNA from various archaeological contexts, reveals a widespread dispersal and use of T. cacao by people in
Amazonian and Pacific coast regions as well as the use of its wild relative species. In addition to recovering T.
cacao aDNA, we also identified the presence of methylxanthine compounds, characteristic of modern T. cacao
seeds, on ancient artifacts from several archaeological sites. Farmers currently face many threats to T. cacao. A
better knowledge of the complex history of cacao domestication, which led to the adaptation of cocoa trees to
their new environments and to their genetic mixing, at the basis of current cacao tree populations, will help to
improve our breeding strategies.

Results
A total of 378 ceramic residues were collected from 352 archaeological items (Table 1, Supplementary Table 1)
representing 19 ancient human cultures from six countries, widespread on the Pacific coast of Ecuador and
Colombia, in Amazonia, and in Central America (Fig. 1 and Table 1). Methylxanthine analyses were carried
out for 326 archaeological items, and aDNA analyses were carried out for 157 archaeological items for which it
was possible to construct and sequence a library. A detailed description of the archaeological items analyzed for
the presence of methylxanthines or of ancient DNA of Theobroma or Herrania, a genus close to Theobroma and
consumed by local populations, is reported in Supplementary Table 1.

Methylxanthine analyses
T. cacao seeds are rich in two methylxanthines, theobromine and caffeine, and have a smaller amount of a third
compound: ­theophylline26. Other South American plant species, such as mate (Ilex paraguariensis), Guarana
(Paullinia cupana) and Theobroma bicolor26–28 also contain these compounds, so the methylxanthine presence
in ceramic residues could be also indicative of the use of these plants in addition to T. cacao. However, the other
species within the Theobroma (or Herrania) genus have much lower levels of theobromine and theophylline; so,
the presence of high levels of these substances in the ceramics would strongly indicate the presence of T. cacao
rather than other Theobroma or Herrania species.
We tested the presence of methylxanthines in the residues of 326 ceramic items, as well as with associated
controls containing (positive control) or not containing (negative control) methylxanthines (Fig. 2 and
Supplementary Fig. 1). Previous studies have noted that environmental contamination can occur during museum
storage, and that caffeine and related methylxanthines can comprise one component of the particulate matter
that can settled on artifacts in ­museums29,30. Methylxanthine contamination also may be due to human activity
during excavations, and by exposure to water and/or micro-organisms29.
To avoid false positive results linked with possible contamination, we used the distribution of methylxanthine
amounts (theobromine, theophylline and caffeine values) (supplementary Fig. 2), to define a threshold of 700pg/
sample to consider a sample as positive for the methylxanthine presence (see material and methods).

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Number
Number of Number of of items
Total number of Source Number of items Number of archaeological archaeological Number of Number of Number of with
Associated archaeological excavation analyzed for both archaeological items with items with archaeological archaeologi cal items with T. Herrania
cultural items analized for (AE) or methylxanthines and items analyzed for theobromine theophylline items with caffeine items analyzed cacao aDNA aDNA

(2024) 14:2972 |
Culture chronology methylxanthines museum (MS) aDNA methylxanthines presence presence presence for aDNA presence presence
Amazonas 350 BC–AD 1200 6 AE 5 6 6 0 2 5 1 0
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Araracuara AD 805–1610 13 AE 11 13 7 4 11 11 3 0
Barlovento 1560–1030 BC 6 AE 3 6 3 0 6 3 4 2
Calima-Ilama 1600 -100 BC 25 MS 12 24 14 4 20 13 12 3
Puerto Hormiga 3090–2552 BC 26 AE 12 26 20 1 26 12 10 8
San Augustin 1000 BC–AD 1 2 MS 2 2 2 1 2 2 2 1
San Jacinto 3750–2000 BC 10 AE 10 10 10 3 10 10 4 0
Zenu 200 BC–AD 1600 1 MS 1 1 1 0 1 1 1 0
Tumaco—La
700 BC–AD 400 35 AE and MS 2 35 4 0 7 2 1 1
Tolita
400 BC– AD
Atacames 2 MS 1 2 1 0 1 1 1 0
1530
Bahía 500 BC–AD 650 15 MS 0 15 1 0 1 0 NA NA
Chorrera 1000–350 BC 33 MS 8 33 5 3 3 8 7 2
Chorrera-Bahia 600–500 BC 2 MS 0 2 0 0 0 0 NA NA
Chorrera-Jama
600–350 BC 1 MS 0 1 0 0 0 0 NA NA
Coaque
Jama Coaque 350 BC–AD 1532 26 MS 2 26 4 1 3 2 2 11

https://doi.org/10.1038/s41598-024-53010-6
Machalilla 1600–1000 BC 6 MS 5 6 3 0 3 5 5 2
Valdivia 3900–1400 BC 96 AE and MS 41 96 35 6 27 41 19 6
Marañon 1882–1642 BC 7 AE 2 7 0 0 0 2 2 1
Total South
312 117 311 116 23 123 118 73 27
America
Olmec 1800–1000 BC 15 AE 0 0 NA NA NA 15 11 1
Maya 600 BC–AD 250 20 AE 10 10 1 1 1 19 11 3
Panama 1500 BC–AD 600 5 MS 5 5 4 4 5 5 4 0
Total Central
40 15 15 5 5 5 39 26 4
America
Total samples 132 133 326 121 28 129 157 99 31

Table 1.  Number of positive archaeological items for the presence of methylxanthine and ancient DNA (aDNA) per associated cultures. The number of archaeological items for which it was
possible to construct and sequence a library is indicated as “Number of archaeological items analyzed for aDNA” Methylxanthines are considered as positive with a value > 700pg/sample.
Presence of T. cacao (or Herrania) sequences is considered as positive for samples showing at least five different sequences for which the first hit is T. cacao (or Herrania) after blast against the
NCBI NT international database. BC: years before Christ; AD: years after Christ.

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Figure 1.  Geographical localizations of genetic groups and human pre-Columbian cultures associated to
the archaeological items analyzed. The eleven genetic groups of the T. cacao species, as previously r­ eported4,
are indicated at the right top of the figure and their native areas localized in the map with the corresponding
colors. Stars represent the area of cultivation of the three old varieties: Criollo (red), Amelonado (light blue)
and Nacional (yellow), and with their corresponding wild genetic group, except for Criollo, for which there is
no very close wild-type genetic group. The approximate sites of occupation of human pre-Columbian cultures
associated to archaeological items investigated in this study are represented on the map by a number and an
arrow: (1) Marañon, (2) Valdivia, (3) Machalilla, (4) Bahia/Chorrera, (5) Jama Coaque, (6) La Tolita/Atacames/
Nariño, (7) San Augustin, (8) Calima Ilama, (9) Amazonas, (10) Araracuara, (11) Puerto Hormiga, (12) San
Jacinto, (13) Barlovento, (14) Zenu, (15) Panama, (16), Maya, (17) Olmec.

Among the 311 archaeological items analyzed from South America, 116 (37%) were positive for theobromine
presence, 23 (7%) for theophylline presence and 123 (39%) for caffeine presence (Table 1). Theobromine and
caffeine presence were observed in archaeological items from all South American cultures except for the seven
Marañon culture items (Fig. 2, Supplementary Table 2).

Ancient DNA analyses

The ancient DNA is known to be highly degraded, characterized by scarcity and damage due to postmortem
­deamination31–33. We adapted the experimental conditions to aDNA post-mortem decay for the first steps of
aDNA analyses (extraction, construction of libraries), to avoid contaminations by modern DNA (see material
and methods, Laboratory environment). We analyzed 157 archaeological items for the presence of T. cacao or
wild relatives, associated with several negative controls.

Identification of T. cacao/Herrania specific sequences

After library construction and sequencing, 13.620 billion of useful pair sequences (with quality scores and
allowing to construct a consensus sequence) were produced. Ceramic residues contain a mixture of DNA from
several organisms and T. cacao or Herrania specific sequences were identified after applying two successive filters:
from the whole set of sequences, a total of 618,048 sequences could first be mapped on the T. cacao genome
(0.0045%). Then, these sequences were BLASTed on the NCBI NT international database. Among these, 19,836
sequences (3.21%) were identified as “first hit” T. cacao specific sequences, and 1,059 sequences (0.18%) were
identified as first hit Herrania specific sequences (Supplementary Table 2).
Of the 157 samples analyzed for aDNA, 99 were positive for T. cacao specific sequences and 31 for Herrania
specific sequences. Among the 118 ceramic samples from South America, 73 had T. cacao and 27 had Herrania
aDNA specific sequences. As for methylxanthine presence, T. cacao specific sequences were observed in all South
American cultures associated with ceramic items analyzed in this work (Table 1, Fig. 3).
None of our negative controls contained ancient T. cacao or Herrania DNA sequences above our threshold
set at five specific sequences.

Ancient DNA authentication

Typical postmortem DNA damages, as previously ­described23, were observed: (1) a high aDNA fragmentation for
all analyzed samples whose mean length of aDNA fragments is 81,65 bp (Supplementary Fig. 3); (2) a decreased
PCR (polymerase chain reaction) amplification intensity when increasing the length of amplified DNA fragments
as observed in all samples positive for cacao presence and reported in Supplementary Fig. 3 for a part of them;
and (3) other postmortem damages, typical of aDNA were also observed, as an enrichment of purines (A and

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Figure 2.  Box plots statistical representation of theobromine and caffeine values (pg/sample) observed in
several Colombian and Ecuadorian cultures. The points in the graphics represent the individual values, and
the numbers in parentheses correspond to the number of samples analyzed per culture. The central box is
delimited by the first quartile (value below which 25% of the data falls when arranged in ascending order) and
the third quartile (value below which 75% of the data arranged in ascending order lies), and with the median
(middle value of the set of values with half of the values less than the median and half the values greater than the
median). The ends of the whiskers are calculated using 1.5 times the interquartile space (the distance between
the 1st and 3rd quartiles), and with outliers drawn over the upper limit. The dashed lines indicate the threshold
at which methylxanthine values are considered positive (700 pg/sample). The number of analyzed archaeological
samples per human pre-Columbian culture is indicated in parentheses.

G) and a higher C– > T substitutions around the ends of aDNA fragments, due to cytosine deamination, as
represented in Supplementary Fig. 4 for two samples.

Relationships between aDNA and methylxanthine presences

The presence of theobromine was mostly our first selection criterion to select the samples to be analyzed for
aDNA. We focused on the 120 archaeological items analyzed for both methylxanthines and ancient DNA, and
positive for at least one of them, as reported in Supplementary Table 2. Among them, 96 items are positive for
theobromine presence, regardless of the presence or not of caffeine or theophylline. Among these 96 items,
62 (65%) were positive for T. cacao aDNA presence. If we consider the 26 items positive for the presence of
theophylline, 21 of them (81%) contain also T. cacao aDNA. Similarly, in the same set of 120 archaeological items,
81 items are positive for caffeine presence, and 62 of them (76%) are also positive for T. cacao aDNA presence.

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Figure 3.  Examples of archaeological ceramic items in which we detected T. cacao ancient DNA presence. Item
names and their associated culture: A: P236: sherd from Puerto Hormiga; B: P247: sherd from San Jacinto; C:
P175: ceramic item of the Jaen Archaeological site directly collected, Marañon; D: P325: pot, Valdivia phase III;
E: P345: pot, Valdivia phase II; F: P348: pot, Machalilla; G: P350: Machalilla; H: P57: effigy vessel of a pregnant
woman, Chorrera; I: P137: bottle, Chorrera; J: P306: effigy vessel of an owl, Jama Coaque; K: P307: effigy vessel
of mythic being, Jama Coaque; L: P213: vase, Calima Ilama; M: P214: vase, Calima Ilama.

Thus, a high percentage of T. cacao presence can be predicted by the analysis of either of these methylxanthine
measurements despite their presence in several other South American plants. Among the same set of 120
archaeological items, if we consider only the 85 items positive for cacao aDNA presence, 62 of them (73%) are
positive for theobromine, 62 (73%) are positive for caffeine and 21 (25%) are positive for theophylline. The lower
success rate for the latter is probably explained by the much lower theophylline levels (5000 to 10,000 times
less) existing in cacao ­beans26, making it a less sensitive predictor for the presence of T. cacao in the ceramics.

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Diversity and ancestries of cacao ancient DNA

To identify the genetic origins of the T. cacao aDNA present in the different archaeological cultures studied
here, we tried to elucidate their genetic ancestry and to link them to possible routes of introduction of T. cacao
from Amazonia. To this end, we selected a reference set of 76 modern accessions, recently re-sequenced25,
representative of the diversity of the T. cacao species. We established a phylogenetic tree with 460 SNPs
(Supplementary Fig. 5), confirming previous results that show T. cacao’s structure in eleven genetic g­ roups4.
This phylogenetic tree highlights and confirms the closest genetic distance between Criollo and the new
Caqueta genetic group located in Southern Colombia (Fig. 1), a region geographically close to the Araracuara
archaeological ­site34. It also confirms the close relationships between some modern Nacional ­ancestors19 and
genotypes of the Nacional group located close to the Palanda archaeological site (PAL) in the southeastern
Ecuador.
The phylogenetic tree also confirms the low genetic distance between all the Guyanese and Peruvian genetic
groups—Marañon, Guiana, Iquitos, Nanay, ­Amelonado2,4. The Curaray group appears close to the Criollo group
in accordance with previous r­ esults2. This phylogenetic tree also displays a close genetic distance between the
Nacional and Contamana groups contrary to previous results obtained with microsatellite ­markers4 or gene
presence and absence variations (PAV) ­markers25. Overall, these 76 accessions provide a good representation
of the currently known T. cacao diversity and genetic groups, making them suitable candidates to analyse the
ancestry of ancient DNA sequences. We also added five representatives of wild relative species to our analysis,
to identify the possible consumption, in ancient times, of species close to T. cacao.
As reported in Table 1, 157 archaeological items could be analyzed for the presence of T. cacao/Herrania
ancient DNA (independently of methylxanthines analyses). Among them, 99 contained aDNA from T. cacao.
After SNP extraction of these sequences, common to both the reference collection and each of the archaeological
samples, structure genetic analyses could be carried out on 61 archaeological items and genetic distance analyses
carried out on 66 of them.
All detailed geographic origin information for these samples is gathered in Supplementary Table 1. A variable
number of SNPs common to the aDNA and the reference collection (Supplementary Table 3) can be observed,
but all items had a minimum of 20 common SNP markers. Each aDNA sample was analyzed individually with
the reference collection using their common set of SNP markers. We first analyzed the structure of the samples
and estimated the genotype membership proportions of T. cacao genetic groups and wild relatives for each
archaeological item, using Structure s­ oftware35 (Supplementary Table 3, Supplementary Fig. 6 and Fig. 4). We
identified a high degree of diversity of cacao ancestries among the human cultural groups. In the Valdivia
culture, we found samples related to the Marañon and Contamana genetic groups, suggesting interactions with
the Peruvian region where these T. cacao groups originated, but also samples related to Nacional, Criollo and
Amelonado groups. Plots structure, at different values of K is reported as examples for two Valdivia items,
having important Amalonado, Criollo and Nacional ancestries, confirming these ancestries at different values
of K (Supplementary Fig. 7). A similar diversity of origins was found in more recent Ecuadorian cultures such as
Machalilla and Chorrera, with cacao samples displaying genetic structures that are either completely Nacional,
or hybrids with Nacional mixed with other types, including Amelonado, as is observed presently in the modern
Nacional variety. The samples from the Marañon culture in Amazonian Perú, show the presence of Criollo and
Curaray genetic groups.
A similar range of cacao genetic diversity can also be observed in ancient cacao samples from Colombia, with
some samples highly related to Criollo observed in samples from Puerto Hormiga and Calima-Ilama cultures.
Likewise, we observed diverse genetic origins of ancient cacao samples from Olmec and Maya sites in Central
America. We found that, contrary to previous research, Criollo was not the only variety consumed. Instead, we
found evidence that Amelonado, Nacional and/or Iquitos genetic groups were present by the Olmec period. The
Nei genetic distances calculated between the ancient DNA sequences and the modern genetic groups confirmed
the results of structure analyses (Supplementary Table 4).
We also found that wild species of Theobroma or Herrania genus were consumed by nearly all cultures that we
analyzed. In a few cases, such as some samples from the Barlovento, Chorrera, and Leticia sites, it was possible
to identify a Herrania species used by human populations. Theobroma speciosa was also identified in a sample
from a Calima Ilama item.

Discussion
Recent findings document the domestication of T. cacao in the Ecuadorian Amazon region, its region of origin,
by at least 5300 years ­ago18. Our new findings demonstrate the large landscape of domestication of cacao, out
of its area of origin, along the Pacific coast of South America, occurring concurrently during this same early
time period and in subsequent time periods. These new findings are revealed by the presence of T. cacao ancient
DNA on almost 30% of the ceramic items that we tested, that were used in both domestic and ritual activities.
The wide range of ceramic artifacts containing evidence of ancient T. cacao DNA, demonstrates the extensive
and ongoing use of both T. cacao and its wild relatives by ancient peoples who lived along the Pacific coast of
northern South America.
Theobroma cacao originated in Amazonia and therefore its presence along the Pacific coast, reveals past
interactions between Amazonian peoples and their neighbors to the west along the coast. These interactions
may have included both human migration and trade exchanges that encouraged the dispersal of cultivated
plants along with a host of other trade ­items36. Several authors have reported intense commercial exchanges
among Amazonian ­regions37,38 all along the vast river networks throughout Amazonia. Goods, plants, and
people could have travelled up to 1000 km, exchanging complementary tools, foods and other materials needed
for people’s livelihoods, as well as knowledge and ideas that were an essential part of Amazonian c­ osmology39,40.

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Ecuador Valdivia 3900 - 1400 BC Criollo Caqueta Curaray

P345 (26) P318 (33) P449 (26) P319 (48) P325 (175) Nacional Purus Contamana
Marañon Iquitos Amelonado
Nanay Guiana Wild relative sp.
II IIa III III
P329 (84) P334 (152) P342 (90) P343 (60) P344 (69) P340 (237)
Colombia
P238 (43) P226 (38) P227 (42) P230 (33) P234 (41) Puerto
III III III VIII VIII
Hormiga
P347 (38) P348 (47) P129 (95)
3090 - 2552 BC
Machalilla
1600 - 1000 BC P213 (35) P214 (99) P287 (21) P288 (47) P300 (40)
Calima - Ilama
1600 - 100 BC
P57 (45) P128 (22) P132 (55) P137 (64) P148 (44)
Chorrera
1000 - P200 (269)
P201 (35)
350 BC San Augustin
1000 BC – AD 1 Zenu
P49 (30)
Jama Coaque P111 (122) La Tolita 200 BC – AD 1600
350 BC – AD 1532 600 BC – AD 400 P271 (20)

Amazonas
P104 (54)
Atacames 350 BC - AD 1200
400 BC – AD 1530
Peru
M125 (64) M145 (21) M152 (27) M97 (262)
PER173 (27) PER175 (22)
Marañon
Mexico M110 (299)
Olmec
1800 -
1880 - 1640 BC
1000 BC

P501 (30) P506 (39) P508 (25) P513 (26) GR65 (65)
P285 (58)
Belize Maya
Panama Panama 600 BC -
1500 BC – AD 600 AD 250

Figure 4.  Visualization of the genotype membership proportions of T. cacao genetic groups and wild
relatives for each archaeological item. The genetic structure of each archaeological item residues was analyzed
individually with the reference collection. The Valdivia phase is indicated at the right and bottom side for each
Valdivia sample when determined. The structure of T. cacao ancient DNA sequences from ceramic residues
was determined using a Bayesian model-based clustering method, implemented in the STRU​CTU​RE software
V2.3.435. The genotype membership proportion was calculated using a set of at least 20 SNP markers, common
to the sample and to the reference collection and is indicated by colours corresponding to the genetic groups
mentioned at the top right side of the figure. Only archaeological items with unambiguous ancestry values
between two groups are reported in this figure. The complete ancestry values are reported for all analyzed items
in Supplementary Table 3. The number of SNP used for each Structure analysis is indicated in parentheses.

Amazonia was a major world center of plant domestication, where selection began in the Late Pleistocene to
Early ­Holocene41,42, thus, exploiting and generating a new diversity provided by a genetic mixing of introduced
T. cacao trees from different origins. Within Amazonia, an important center of resources was reported in the
Iquitos region of Perú41, where several T. cacao genetic groups originated: most notably the Marañon, Nanay,
Iquitos and Contamana groups. In Colombia, an independent center of domestication was r­ eported43,44, and
a study of plant food production in Colombian tropical forests reported the adoption of exogenous plants
domesticates, as manioc and maize, as early as the middle H ­ olocene45. Interactions between Amazonia and the
Pacific coastal peoples that involved the use and domestication of T. cacao likely occurred during the earliest
stages of agriculture. This inference is based on our observation that cacao originating from several T. cacao
genetic groups located in the Peruvian Amazonia, was observed in the oldest Pacific coast cultures of Valdivia, in
Ecuador, and Puerto Hormiga and San Jacinto in Colombia, dating to more than 5,000 years ago. In samples from
the Valdivia culture sites, dating to Phase III (2950-2600 BC), the presence of T. cacao genotypes originating from
the Peruvian Marañon and Nanay groups suggests that people in this region had early long-standing contacts
with the Peruvian Amazon. Genotypes related to the Nacional group were also observed in the Valdivia ceramic
residues. This genetic group is located in southeastern Ecuador, where the Mayo-Chinchipe-Marañon culture
existed contemporaneously with the coastal Valdivia culture. Mobility was one of the characteristics of the Mayo
Chinchipe-Marañon people. They navigated the many riverine tributaries that flow into the main channel of the
Amazon River, thus allowing the rapid and extensive spread of plants (including T. cacao) and other products,
throughout this vast ­region46,47. A similar situation is encountered for the samples originated from the Caribbean
coast of Colombia (Puerto Hormiga and San Jacinto) where cacao genotypes related to the Marañon, Contamana

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and Iquitos genetic groups, originating in Perú, were observed, reflecting direct or indirect early contacts with
the Peruvian Amazon. Even if modern-day diversity has changed over millennia, the concordant ancestry and
parentage results obtained by analyses of genetic structures and genetic distances support the probable origin
we identified and the genetic mixing of cacao trees introduced to the Pacific coast.
Theobroma cacao was also introduced to Central America, but its introduction from the Amazon, either
northward overland, or by sea along the Pacific coast, still raises many questions. Several authors have suggested
exchanges between the Pacific coast of Ecuador and Mesoamerica, based on many similarities between ceramics
of the Pacific coast of Ecuador and the Pacific coast of G ­ uatemala37,48–50. More recently the evidence for pre-
Columbian maritime long-distance contacts between western Mexico and the Pacific coast of northwest South
America were synthesized and r­ eassessed51, considering an area spanning 4000 km of coastline and over 4000
years of interaction. Early contacts could be highlighted: the observation of a ceramic motif of the early formative
Valdivia VI phase (2200-2000 BC) during the Mesoamerican Early Formative P ­ eriod52, evinces early long-
17
distance Pacific coastal interactions. Several other facts have been pointed o­ ut showing that maritime navigation
was possible at early times and could have supported cacao’s dispersal from Ecuador to Mesoamerica through
vast interconnected political-economic networks. Recent findings based on ancient DNA analyses have shown an
early dispersal of maize, first domesticated in Mexico, to Perú 6700–5000 cal years BP (Before Present), through
a rapid coastal migration route from the Pacific l­owlands53. These findings demonstrate the early and possible
rapid exchange of plants between Mesoamerica and the Pacific coast of South America where the Pacific coast
of Ecuador and cacao may also have been involved in these exchanges.
Our archaeogenomic results have shown the diversity of the genetic origins of the cacao varieties consumed
by ancient peoples, and call into question the previously proposed patterns of the introduction of cacao trees
on the Pacific coast of Ecuador and in Central America. The three ancestors of the modern Nacional variety—
Criollo, Amelonado, Nacional—already existed on the Pacific coast during Valdivia times, contrary to what we
had previously thought. The present study also suggests that the domestication of the other fine flavor variety,
the Criollo variety, is likely to be older than previously thought. While Criollo’s origin remains unknown, it
shows a greater proximity to the Caqueta genetic group, located in Colombian A ­ mazonia4. Often hybridized
with other genetic groups, it is observed in archaeological samples from some of the oldest cultures in Ecuador
(Valdivia) and Colombia (Puerto Hormiga, Calima Ilama). Criollo is characterized by many private a­ lleles4,
suggesting that the Criollo group probably results from a long domestication process, for which a loss of genes
has been recently characterized by a pangenome study showing that Criollo has the fewest genes compared to
other genetic ­groups25. Criollo was supposed to be the unique variety cultivated in Central America during the
Olmec and Maya p ­ eriods7,14,15. However, our results indicate that this was not the case. Criollo, along with other
genetic groups from different regions were all present in ancient Central American cultures. Among these,
the main ones were the Nacional and Amelonado genetic groups, respectively originating from southeastern
Ecuador, and northern Brazil.
These results have shown the complex and early history of cacao domestication and suggest that it was linked
to long-distance trade and exchange patterns that started at least at mid Holocene times. They also demonstrate
the effectiveness of archaeogenomic approaches in tracing plant domestication histories. Ultimately, cacao’s long
history is intimately intertwined with the diversity of new geographic environments and cultural groups where
it has thrived and evolved, with intense gene-flows between remote T. cacao populations and the emergence of
hybrid forms, favorable to their adaptation to new environments and adoption by local human cultures.
The multi-billion dollars cacao industry supports the economy of many countries and cacao is one of the
world’s most important cash crops. However, T. cacao is subject to many threats, including the susceptibility to
disease and climate change. Our multidisciplinary approach, involving archaeogenomics, genomics, archeology
and biochemistry is a key approach to unraveling the complex ancestry of T. cacao underlying today’s cacao
populations. It will help to better manage and exploit genetic resources in order to deal with these threats.

Material and methods

Cultural provenance of the archaeological items analyzed
Residues from the interior walls of ceramics were collected from archaeological items stored in museums or
coming from direct excavations. Residues were collected from a total of 352 items, from 19 pre-Columbian
cultures widespread in South America (Ecuador, Colombia, Perú), and in Central America (México, Belize,
Panama). These cultures span about 5000 years and are mostly scattered along the Pacific coast. (Fig. 1 and
Supplementary Table 1).

Collection of ceramic residues

No conserved cacao organs or cacao seed fragments were found on the ceramics. Only traces of putative charred
food residues adhering to the interior of the ceramics walls or sherds could be collected.
Most of the collection of ceramic food residues were made using rayon swabs (Copan-Italia) saturated with
extraction buffer (0.1 M Tris, 0.45 M EDTA and 0.25 mg/ml proteinase K)54. The swabs were wiped against the
inner walls of the ceramics and stored in their sterile box at 4°C until returning to the CIRAD-AGAP laboratory
(Montpellier-France), where all samples were stored at -20°C. About 10 swabs were collected per ceramic item.
For the Maya and Olmec samples, the interior surface of each ceramic vessel or sherd was lightly scraped using
a new piece of fine-grained sandpaper, and the powder conserved for further analyses.

Methylxanthine analyses
Three methylxanthine components present in T. cacao were analyzed: theobromine, theophylline and caffeine.
Either one rayon swab or 0.5 g of powder was used for analyses of the archaeological items.

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Extraction procedure and biochemical analyses

The analyses were made in two different laboratories:

1. The laboratory of biochemistry from Gaikwad Steroidomics Lab LLC, Davis (USA).
­ escribed18.
  Extraction procedure and biochemical analyses made in this lab were carried out as previously d
2. The « laboratoire de mesures physiques », Institut des biomolécules Max Mousseron, Université de
Montpellier (France).

Extraction procedure was slightly different: Samples were incubated with 600 μl milli-Q water at pH3, at 80 °C
for 30 min. After incubation, samples were vortexed and centrifuged. the supernatant was transferred in a 35
µm Macherey filter column and centrifuged at 10,000 g for 10 mn. The supernatant was desiccated by speedvac,
a vacuum concentration system, and then diluted in 10 µl of milli-Q water.
UPLC-MS/MS (Ultra-Performance Liquid Chromatography—Mass Spectrometry) analyses were carried out
according to the following protocol:
Theobromine, theobromine-d6, theophylline and caffeine reference standards were purchased from Sigma-
Aldrich. Acetonitrile, methanol and formic acid were ULC/MS grade and the water was milli-Q grade. The
analyses were recorded on a LC–MS 8050 triple quadrupole (Shimadzu) coupled to a Nexera chromatographic
chain (Shimadzu). Positive mode electrospray ionization was used, with interface voltage at 4kV, nebulizing gas
flow of 3L/min, heating gas flow of 10 L/min, interface temperature of 300 °C, a DL temperature of 250 °C, a heat
block temperature of 400°C and a drying gas flow of 10 L/min. The transitions and the collision energies were
optimized for each compound: 181.10 → 138.20 with collision energy of -18 eV for theobromine, 181.10 → 124.15
with collision energy of -18 eV for theophylline, 195.00 → 138.20 with collision energy of -20 eV for caffeine and
187.20 → 144.15 with collision energy of -18 eV for theobromine-d6.
Before the analyses in LCMS, the samples were passed over C18 ODS 100mg Solid Phase Extraction (SPE)
(Agilent). For this, the columns were conditioned with 1 mL of methanol, followed by 1 mL of water. The samples
were taken up in 100 µL of water acidified with 1% formic acid before being placed on the columns.
Then 1 ml of water was passed through. The products of interest were unhooked from the column by passing
1 ml of methanol followed by 0.5 ml of isopropanol. For each sample, these two solvents were combined and
dried. The samples were taken up in 20 µl of solution of theobromine-d6 at 5 ng/mL and 10 µl were injected
during the LCMS analysis.
Analytical separations on the UPLC system were conducted using an Acquity UPLC BEH C18 1.7 μm column
(50 × 2;1 mm) at a flow rate of 0.6 ml/min. The temperature of the column oven was 40°C. The gradient was
started with 100% A (0.1% formic acid in H ­ 2O) and 0% B (0.1% formic acid in C
­ H3CN). Then the percentage
of eluent B was increased linearly to be at 14% at 0.7 min. We remained on this plateau until 1.25 min, before
increasing the percentage of B linearly to 50% at 1.5 min, then to 100% B at 3 min. The run time was 5 min. The
data obtained after LCMS coupling were processed by the Labsolution Insight software (Shimadzu).
For each compound, standard solutions at 1mg/ml were prepared. Theobromine-d6 was used as an internal
standard. From these stock solutions, mixtures grouping the compounds theobromine, theophylline, and caffeine
at different concentrations (1; 2; 4; 6; 8; 10 and 15 ng/ml) and theobromine-d6 at a concentration of 5 ng/ml were
used as calibration solutions. Two other mixtures of compounds were made from new weightings to create quality
control (QC) solutions at concentrations of 1.5 and 9 ng/ml. The QCs were injected with the standards to validate
the calibration line. A solution consisting only of the internal standard was used as a blank as well as negative
controls for which rayon swabs, not wiped against the inner walls of the ceramics, were analyzed following
the same protocol of extraction and UPLC-MS/MS analyses. The retention times obtained were 1.05 min for
theobromine and theobromine-d6, 1.38 min for theophylline and 1.70 min for caffeine. From the concentrations
obtained and the sample recovery volume, the quantities in pg of the various compounds were calculated.

Determination of methylxanthine threshold

Ideally, the threshold for a positive presence of methylxanthines depends on the Limit of Detection (LOD),
where we can observe a signal for the analyte of interest, but too small to be measured accurately, and on the
Limit of Quantitation (LOQ) of the analyte when no signal is observed. This is determined by the “signal” to
“noise” ratio. However, possible airborne contamination during storage of archaeological items in the museum
reserves was already ­reported28,29 and could increase the values. To prevent false positive results, we established
methylxanthine values distribution (Supplementary Fig. 2) to estimate the possible environmental background
linked to these possible levels of contamination and establish a threshold for the detection of positive samples.
After plotting the distribution of methylxanthine amounts (theobromine, theophylline and caffeine values)
observed in all samples (Supplementary Fig. 2), we observed a clear break in these distributions around 200 pg/
sample for each series of methylxanthine values. The high number of values < 200 pg/sample may correspond to
the ambient contamination already d ­ escribed29. Thus, to be conservative we chose a threshold of 700 pg/sample
and considered a sample as positive where the methylxanthine amount was > 700 pg. All values < 700 pg were
considered and reported as “0” in Supplementary Table 2.
We also ran positive and negative controls (Supplementary Fig. 1). In these analyses all negative controls
were < 250 pg/sample.

Laboratory environment for ancient DNA analyses

The ancient DNA is highly degraded, characterized by scarcity and damage due to post-mortem decay and
­deamination31. We adapted the experimental conditions to prevent contamination by modern DNA, preferentially
amplified during PCR steps: all pre-PCR experiments were conducted under sterile conditions in the platform

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“Paléogénomique et génétique moléculaire” (P2GM) of the French “Muséum National d’Histoire Naturelle” at
the “Musée de l’Homme” (Paris). This laboratory is dedicated to ancient DNA analyses mainly conducted on
humans and animals and there has never been previous work on Theobroma species. It is equipped with positive
high-pressure air system, with continuous filtering of incoming air, daily UV light irradiation, laminar flow hoods
with HEPA filters, and all surfaces frequently cleaned. The experimenters wore whole-body protective clothing
including gloves and shoe protection.

Extraction of ancient DNA

Ancient DNA extraction from ceramic wall was first carried out on ­amphoras55. The protocol used to extract T.
cacao ancient DNA from ceramic items collected in the South Amazonian r­ egion18, were adapted in this work
as follow:
Four swabs were used for each aDNA extraction made from ceramic residues. Ancient DNA extraction was
made with the Qiagen, DNeasy PowerLyzer PowerSoil Kit, which effectively removes PCR inhibitors such as
humic acids, and according to the described manufacturer’s procedure except for the binding step for which
the concentration of the C4 saline solution was increased 1.6 times to retain the smallest fragments. One or
two independent extractions (made for 30 archaeological samples) were made for each archaeological sample,
named MX.
Several negative controls were processed alongside the ancient samples.

Libraries construction
Construction of the libraries using the NEXTflexTM Rapid DNA-Sequencing Kit (Bioo Scientific) were carried
out for MX1 to MX12 according to the protocol indicated in the kit.
Dual-indexed Illumina sequencing libraries were prepared for MX13 to MX232 using three main r­ eactions56:
blunt end-repair, adapter ligation, and nick fill-in reaction as reported in supplementary methods.

Sequencing steps
All samples were sequenced in paired ends (2 × 150 pb) on the iGenSeq core facility of ICM (Institut du Cerveau
et de la Moelle Epinière—Paris). Each run was performed on ILLUMINA NOVASEQ 6000 with 300 cycles
cartridge (150PE). Cartridge was choose depending of the number of samples to obtain at least 2*30 Millions of
150 bases reads per sample. Bcl files were converted in fastq format with ILLUMINA bcl2fastq.
At the beginning of the experiments, additional sequencing of aDNA samples (MX1 to MX12) were made
on the Montpellier GenomiX (MGX) platform (Montpellier-France).
The aDNA libraries were sequenced according two possible different strategies reported for each sample in
Supplementary Table 1: or directly by whole genome sequencing (WGS) or after a step of Targeted capture (TC)
as described ­previously18. The targeted captures were carried out with a custom-designed Mybaits sequence
capture kit (V 3.02) provided by the Microarray company and defined from 4847 unique nuclear Theobroma cacao
sequences containing SNP sites. These SNP were identified by GBS (genotyping by sequencing) on a collection
of Theobroma and Herrania genetic resources, and are located in genes for about 70% of them. The pipeline used
­ escribed25.
for filtering, demultiplexing, and processing the reads is already d

Bioinformatic sequences treatment

The food ceramic residues contain ancient DNA from a mixture of several species of bacteria, fungi, plants, and
animals, and often with many similarities between sequences from different species. Thus, filters were necessary
to specifically identify the T. cacao sequences. A first filter was the mapping against the T. cacao genome and a
second filter was necessary to identify the sequences specific to T. cacao through a BLAST against data sequences
from the NT NCBI international database.
To apply such a selection, sequencing adapters and low quality nucleotides (quality value < 20) were first
removed from Illumina paired-end reads using Cutadapt v3.457 and Trim Galore v 0.6.6. Reads shorter than
30 nucleotides after trimming were discarded. Then, pairs of reads were merged with FLASH v1.2.1158, using
default settings, and redundancy from the merged DNA fragments was reduced using cd-hit-dup program
(parameter -e 0) from the CD-HIT package v.4.8.159. Then, low complexity/entropy sequences were removed
from the pre-selected aDNA fragments using bbduk program (parameter entropy = 0.7) from BBTools v38.9060.
Resulting sequences were then aligned to the Theobroma cacao reference g­ enome61 with bowtie2 v2.4.262
parameter –very-sensitive) and sequences that aligned at least once to the cacao genome were conserved for
further analysis. Remaining sequences were then searched against the GenBank nucleotide collection NT with
NCBI BLAST + v2.10.163. Blast results were passed to the B ­ ASTA64 taxonomic classification tool v1.3.2.3 and only
sequences with the best Blast Hit being Theobroma cacao or Herrania were kept for further analysis. However,
these filters are very stringent, as only sequences appearing as 1st hit T. cacao were conserved, excluding any T.
cacao DNA sequences homologous to other species after blast on international databases.
The presence of T. cacao (or Herrania) sequence was considered as positive in an archaeological sample when
at least five different T. cacao (or Herrania) first hit sequences were identified in the sample. None of our negative
control had ancient T. cacao or Herrania DNA sequences above this set threshold. We used Krona schemes
to visualize the relative abundances of the several sequences species within the metagenomic classifications,
identified as “first hit” after blast against the NCBI NT ­database65, as represented in Supplementary Fig. 8 for
two archaeological items from the Valdivia and Puerto Hormiga cultures.

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Ancient DNA authentication

To further support the antiquity of the DNA extracted from ceramic residues, typical signatures of post-mortem
DNA ­damage23, such as small size fragments and chemical damages particularly present at the ends of aDNA
fragments were searched for. Pre-selected ancient DNA fragments, obtained after aligning the reads on the T.
cacao genome were processed using MapDamage V2.2.166. Several DNA damages could be analyzed:

Mis‑incorporations observed at the ends of aDNA fragments

An enhanced cytosine deamination observed mostly in 5’ overhanging single strand DNA ends will translate
itself into an excess of cytosine to thymine (C to T) mis-incorporations at 5’ ends of aDNA sequences (and
complementary guanine to adenine (G to A) at 3’ ends when PCR amplified. MapDamage permits evaluation of
these transitions in comparison with the corresponding T. cacao genome sequences.

Purine frequency around the ends of aDNA fragments

Purine (A and G) deamination can result in an enhanced fragmentation of ­DNA23,67,68 and MapDamage allowed
evaluation of the purine frequency around the ends of aDNA fragments.

Evaluation of DNA fragmentation due to post‑mortem damages

Mostly small DNA fragments remain and can be sequenced because of post-mortem d­ epurination67,68, leading to
DNA strand fragmentation. We used MapDamage V2.2.1.66 to display the fragment size distribution as reported
in Supplementary Fig. 3 for all positive samples.

Impact of amplified aDNA fragment size on PCR intensity

In the case of ancient DNA, PCR amplifications with primers amplifying fragments of an increased length will
be effective with a decreased intensity depending on the length of the DNA ­fragments69.
Primers were designed in the mitochondrial Cytochrome C oxidase subunit 2 gene, amplifying DNA fragments
differing in length but having a similar PCR efficiency tested on a modern DNA (Criollo B97-61 genotype)
(Supplementary Table 6): Cyto 66b: 66 bp, Mito-197: 197 bp, Mito-290: 290 bp, Mito-543: 543 bp.
Real-time PCR analysis was carried out in a BioRad CFX96 Touch Real-Time PCR Detection System (Bio-
Rad Laboratoires) using the following steps: 98°C for 3 min; a touchdown PCR [initial 10 cycles; 98°C for 15 s,
58°C for 20s (-1°C every cycle), 72°C for 20s], followed by 50 cycles of 98°C for 15°C, 48°C for 20 s, 72°C for 20s,
then a final step at 72°C for 8 min. To confirm product specificity a melting curve analysis was performed as the
last step. The real-time PCR was carried out in a 10 μl reaction volume containing 5 μl of 2X « SsoFast EvaGreen
Supermix » (Bio-Rad Laboratoires), 0.1 mM of BSA (ref. B9200, New England BioLabs), 500 nM of forward and
reverse primers (Supplementary Table 6), 1 µl of water, and 2 µl of DNA extracts. In each run, blank (negative
control) and a positive control were added.
Data were analyzed using CFX Maestro Software (Bio-Rad Laboratoires) set with default parameters to
determine the cycle threshold (CT), i.e. the number of PCR cycles required for the fluorescent signal to exceed
the background level.

SNP identification
A re-sequencing and pan-genome project of 216 modern T. cacao accessions was recently carried o ­ ut25. The
genomes of 185 modern accessions of T. cacao and five accessions of cacao wild relative species (T. grandiflorum,
T. bicolor, T. speciosa and Herrania nitida) were newly sequenced using the whole genome shotgun Illumina
technology (NCBI Bioproject PRJNA558793). The generated 150 bp paired-end reads were mapped on the V2
cacao reference ­genome61 using Bowtie2 (v. 2.4.2)62. SNPs were then called using NGSEP V. 4.0.070, using filtering
criteria based on genotyping quality (-q 40) and on a minimum read death to keep a genotype call (minRD 5–10)
and a SNP database was established comprising 31,910,149 SNPs. A subset of 76 T. cacao accessions representative
of the genetic diversity of the species and five relative wild species was selected to perform genetic analyses aiming
to identify the ancestry of each archaeological sample residue.
To take into account the potential deaminated DNA damage at the ends of the fragments, we followed the
suggestion to remove the SNP, corresponding to potential C-to-T/G-to-A substitutions, located at the two first
bases of each end of aDNA fragments up to a percentage of 2 to 4% C-to-T substitutions observed at the ends
of aDNA f­ ragments71. In our case, using Mapdamage software, a mean of 4 to 5,5% of C-to-T substitutions
were observed at the ends of aDNA fragments. So, to reduce the risk of false SNP, we remove from the analyses
the putative SNP located at the first five bases of each end of aDNA fragments, and corresponding to potential
C-to-T/G-to-A substitutions only.
To determine the SNP alleles within the ancient DNA fragments, the genomic regions of modern accessions,
homologous to the aDNA sequences, were identified using Blastn, and their SNP extracted, given the known
position, on the cacao genome, of SNP identified in the modern accessions: the aDNA sequences were aligned
with the homologous modern sequence, and its corresponding SNP alleles extracted. Several SNPs could be
identified in a given aDNA sequence.
Only ancient DNA sequences mapping in a unique location on the cacao reference genome were retained for
further genetic analyses. About 3% of ancient DNA fragments for which the SNP allele does not correspond to
any of the two alleles present in the collection of modern accessions, which could be explained by substitutions
due to aDNA degradation, were discarded. Between 20 and 528 SNPs were identified according to the aDNA
samples, with a mean of 74 SNPs per Sample.

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Reference collection
To identify the genetic origin of ancient cacao DNA collected in the ceramic vessels, a reference collection was
selected. This collection includes 76 T. cacao accessions representing the 11 genetic groups recently i­ dentified4:
Criollo (8 ind), Caqueta (8 ind), Curaray (8 ind), Nacional (8 ind), Purus (7 ind), Contamana (4 ind), Marañon
(7 ind), Iquitos (7 ind), Amelonado (4 ind), Nanay (8 ind), Guiana (7 ind).
This collection includes also five accessions from four wild relative species: Herrania nitida (2 ind), Theobroma
grandiflora (1 ind), Theobroma bicolor (1 ind), and Theobroma speciosum (1 ind), (Supplementary Table 5 and
Fig. 1).
A phylogenetic tree was constructed with the Darwin s­ oftware72 for the 76 T. cacao accessions constituting
the reference collection and with a set of 460 SNP markers selected among the SNPs identified in the aDNA
sequences, and widespread in all chromosomes. A distance model, based on the dissimilarity matrix calculated
using the neighbor joining assembly m ­ ethod73, with 500 bootstraps as implemented in Darwin 6.0.14 software,
was used to represent the genetic distance between the 11 genetic groups (Supplementary Fig. 5).

Genetic analyses
Two types of genetic analyses were then carried out for each archaeological sample analyzed individually with
the reference collection:

Structure analyses
We used a Bayesian model-based clustering method, implemented in the STRU​CTU​RE software V2.3.435, to
identify distinct genetic groups in the reference collection (T. cacao or its wild relatives), and to establish the
genotype membership proportion of each genetic group for each archaeological item using the same set of SNP
markers. STRU​CTU​RE was run under an admixture model, using a burn-in initial period of 100,000, a run length
of 300,000 steps, and 10 independent runs for each sample where K values equalled from eight to twelve. Only
archaeological samples with a minimum of 20 SNP markers were considered for these analyses.
The different genetic groups can be dissociated at different values of K, according to the number of SNP
markers revealed in the set of aDNA sequences. We started to carry out the analyses with a maximum of K = 12
corresponding to the known structured modern populations. When the analyses with K = 12 did not allow to
clearly distinguish the components of ancestry of the archaeological item, we continue the analyses, gradually
decreasing the K values. Generally, we obtained this distinction with K values higher that K = 8. For each sample
the higher K value allowing to clearly infer ancestry of archaeological samples to T. cacao genetic groups was
chosen (Fig. 4, Supplementary Table 3, Supplementary Fig. 6).
When it was not possible to differentiate the genotype membership proportion between two given groups,
due to their higher relatedness (Supplementary Fig. 5), as Nanay and Amelonado, and to the number of SNP
used for the analysis, both possible attributions were reported in the Supplementary Table 3.

Genetic distances
With each set of SNP markers identified commonly for each archaeological sample and the reference
­ istances74, adapted to small effective size, were calculated between the ancient DNA
collection, Nei’s genetic d
sequences, selected as specific T. cacao or Herrania sequences, and the other 12 genetic groups (including the
five wild Theobroma and Herrania accessions taken as a separate group) using GENETIX software V4.05.275
(Supplementary Table 4).

Data availability
Raw sequence reads were deposited in the Sequence Read Archive (SRA) of the National Center for Biotechnology
Information (NCBI) (BioProject: PRJNA1049643), and accessible at the following link: https://​www.​ncbi.​nlm.​
nih.​gov/​sra/​PRJNA​10496​43. The sequences specific to T. cacao (aDNA_T_cacao_sequences) and to Herrania
(aDNA_Herrania_sequences) are available in the following websites: https://​cocoa-​genome-​hub.​south​green.​
fr/​sites/​cocoa-​genome-​hub.​south​green.​fr/​files/​downl​oad/​aDNA_​Herra​nia.​fna.​gz. https://​cocoa-​genome-​hub.​
southg​ reen.f​ r/s​ ites/c​ ocoa-g​ enome-h
​ ub.s​ outhg​ reen.f​ r/fi
​ les/d
​ ownlo
​ ad/a​ DNA_T_c​ acao.f​ na.g​ z. The traits of human
cultures associated with archaeological samples are reported in Supplementary Table 1. The other datasets are
available from the corresponding author upon request.

Received: 6 August 2023; Accepted: 25 January 2024

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Acknowledgements
We gratefully acknowledge Yannick Marie and Agnes Rastetter who managed most of the sequencing activities
of this work, which were mostly carried out on the iGenSeq core facility of ICM (Institut du Cerveau et de la
Moelle épinière). We thank the platform MGX-Montpellier GenomiX for the sequencing of the first samples
in this project. We thank the platform “Paléogénomique et génétique moléculaire” (P2GM) of the French
Muséum National d’Histoire Naturelle at the Musée de l’Homme, and the GPTR (grand plateau technique
regional de génotypage) located in CIRAD, Montpellier (France) for support in conducting the molecular
genetics experiments. We thank INPC (Instituto National de Patrimonio Cultural—Ecuador), MAAC (Museo
Antropológico y de Arte Contemporáneo-Ecuador), Musée du quai Branly (France), ICANH (Instituto
Colombiano de Antropología e historia—Colombia), MNC (Museo Nacional de Colombia), UNAM (Universidad
Nacional Autónoma de México) for giving us access to archaeological samples conserved in museum reserves
or during exhibition. We would like to thank the Belize Institute of Archaeology for providing the vessels from
Colha and Pacbitun for residue testing to TGP. A portion of the collection of Valdivia archaeological residues
was conducted when SZ was a Postdoctoral Fellow at the Cotsen Institute of Archaeology, UCLA. We thank CRC
(Cocoa Research Center—Trinidad and Tobago) and CATIE (Centro Agronómico Tropical de Investigación y
Enseñanza- Costa Rica) for providing a part of the modern T. cacao accessions studied in this work. We thank
Céline Bon and Paul Verdu for helpful discussions and comments for the writing of the paper, and Dugane Quon,
Daniel Mezones, Mario Sánchez, Andrés Armijos and Lindsey Paskulin for their help in ceramic residues and
information collects.
We thank the reviewers of this manuscript for their valuable comments which helped to improve it. We thank the
I-Site MUSE and Valrhona for their financial support of this project. This work, part of the MUSE Amazcacao
project, was publicly funded through ANR (the French National Research Agency) under the "Investissement
d’avenir" program with the reference ANR-16-IDEX-0006. We thank the Canadian Tri-Council New Frontiers
in Research-Exploration grant (NFRFE-2018-00066) which has supported the work performed at the University
of British Columbia.

Author contributions
CL, XA, FrV designed research; NA, FR, MGC, TGP, FrV, SQON, FJV, SR, AC, SMR, GSD provided archaeological
samples from excavations or management of museum reserves; CL, XA, RGLS, FrV, NA, TGP, AC, SZ, CS,

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collected archaeological residues; GV, NG performed methylxanthine analyses by UPLC-MS/MS; CL, HV, JU,
OF performed aDNA experiments; CL, BR, XA carried out genetic analyses, CL, FrV, MGC, NA, GV, XA, BR,
JU, AC, TGP, RGLS, SZ, MB contributed to the writing of the paper. All authors reviewed the manuscript.

Competing interests
The authors declare no competing interests.

Additional information
Supplementary Information The online version contains supplementary material available at https://​doi.​org/​
10.​1038/​s41598-​024-​53010-6.
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